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First Report of Drechslera andersenii Causing Leaf Spot on Perennial Ryegrass in Jiangsu China

Hu, J., Yang, J. Y., Li, J., Gao, T., Yang, G. W., Ren, H. Y.
Plant disease 2018 v.102 no.6 pp. 1174
DNA, DNA primers, Drechslera, Lolium perenne, agar, air drying, autoclaving, conidia, cool season grasses, culture media, fungi, glyceraldehyde-3-phosphate dehydrogenase, growth chambers, hyphae, internal transcribed spacers, lawns and turf, leaf spot, leaves, mycelium, plastic bags, polymerase chain reaction, relative humidity, sequence analysis, sodium hypochlorite, solar radiation, streptomycin, sulfates, tetracycline, tissues, China
Perennial ryegrass (Lolium perenne L.) is a cool-season grass that is widely used for overseeding in the transitional zones of China. In June 2017, extensive leaf spots were observed on perennial ryegrass at the campus of Nanjing Agricultural University in Jiangsu province. Lesions were initially 2 to 3 cm in length and 1 to 3 mm in width. As the disease progressed, they expanded and became more numerous, causing a purplish-brown to black stripe with a light brown margin. Finally, large areas of blighted turf developed. Conidia observed on the lesions were 60 to 400 × 8 to 21 μm with 3 to 15 septa (n = 30), and this was consistent with Drechslera andersenii Lam, reported on perennial ryegrass (Lam 1985). Fifteen leaves with lesions were collected and surface sterilized in 0.6% NaClO for 1 min, washed twice with sterile water, air dried, and placed on 1% water agar containing 50 mg/liter of streptomycin sulfate and tetracycline. Plates were incubated in the dark at 25°C for 3 to 5 days. Hyphal tips were transferred to potato dextrose agar (PDA) and incubated at 25°C for 5 days. A fungus with dematiaceous mycelium was consistently isolated. The growth of the mycelium was approximately 2.5 mm per day, and it colonized entire 6-cm PDA plates in 10 days or more. Genomic DNA of a representative isolate was extracted, and the nuclear ribosomal internal transcribed spacer (ITS) region and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS4 and ITS5 (White et al. 1990) and gpd1 and gpd2 (Berbee et al. 1999), respectively. PCR products were sequenced (deposited as MF491854 and MF535178 in GenBank) and showed 99% similarity to the published ITS (AY004805.1) and GAPDH (AY004835.1) sequence of D. andersenii through BLAST analysis. To complete Koch’s postulates, the mycelium of the sequenced isolate was scraped off of 20-day-old PDA cultures, chopped with a sterile scalpel, and suspended with 200 ml of autoclaved water. Two-month-old perennial ryegrass in four plastic pots (15 cm height × 15 cm top diameter × 10 cm bottom diameter) was inoculated with the fungal suspension. Two control pots with perennial ryegrass received equal volumes of sterile water only. All pots were incubated individually in plastic bags in a growth chamber with 16-h daylight at 24°C and 100% relative humidity. Leaf spots were observed 3 to 5 days after inoculation and were prevalent after 7 days. No symptoms developed on control plants. Isolations from symptomatic leaf tissues on PDA produced fungal colonies with morphology consistent with D. andersenii. To our knowledge, this is the first report of D. andersenii causing leaf spot on perennial ryegrass in China.